Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations

Alekseeva, A.A.; Serenko, A.A.; Kargov, I.S.; Savin, S.S.; Kleymenov, S.Y.; Tishkov, V.I.

Авторы: Alekseeva A.A., Serenko A.A., Kargov I.S., Savin S.S., Kleymenov S.Yu, Tishkov V.I.
Журнал: Protein Engineering, Design and Selection
Том: 25
Номер: 11
Год издания: 2012
Издательство: Oxford University Press
Местоположение издательства: United Kingdom
Первая страница: 781
Последняя страница: 788
DOI: 10.1093/protein/gzs084
Аннотация: The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. Based on the alignment of plant FDHs sequences, Asp, Asn, and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in E. coli, purified, and characterized. All amino acid substitutions increased KмHCOO- from 1.5 to 4.1-5.0 mM, whereas the KмNAD+ values remained almost unchanged in the range from 9.1 to 14.0 μM, which is close to wt-SoyFDH (13.3 μM). The catalytic constants for F290N, F290D, and F290S mutants of SoyFDH equaled 2.8, 5.1, and 4.1 s-1, respectively; while that of the wild-type enzyme was 2.9 s-1. The thermal stability of all mutant SoyFDHs was much higher compared to the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N, and F290D introduced into SoyFDH increased the Tm values by 2.9, 4.3, and 7.8oC, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.
Добавил в систему: Тишков Владимир Иванович

	ИСТИНА	Войти в систему Регистрация
	ИПМех РАН
	Главная Поиск Статистика О проекте Помощь

ИСТИНА

ИПМех РАН

Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutationsстатья

Прикрепленные файлы